Beclomethasone Dipropionate

Molecular formula: C28H37CIO7

Molecular weight: 521.1
CAS Registry No.: 5334-09-8 (beclomethasone
dipropionate), 4419-39-0 (beclomethasone)

SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 100 jxL 20 |xg/mL cloprednol + 3 mL ether, shake
10 min, centrifuge at 3000 g, remove the organic phase and evaporate it to dryness under
nitrogen. Take up the residue in 200 |xL mobile phase, inject a 50 jxL aliquot.

HPLCVARIABLES
Column: Nucleosil R 10 C 18
Mobile phase: MeOH: MeCN: water: acetic acid 400:100:200:1
Injection volume: 50
Detector: UV 254

CHROMATOGRAM
Internal standard: cloprednol
Limit of detection: 500 ng/mL

KEYWORDS
plasma
REFERENCE
Wiirthwein, G.; Rohdewald, P. Activation of beclomethasone dipropionate by hydrolysis to beclometha-
sone-17-monopropionate. Biopharm.Drug Dispos., 1990, 11, 381-394

SAMPLE
Matrix: blood, tissue
Sample preparation: Acidify plasma or lung tissue homogenate to pH 2 with 500 mM
HCl, add 100 |xL 20 |JLg/mL IS, extract with 8 mL dichloromethane. Evaporate the organic
layer to dryness under vacuum, reconstitute in 120 |JLL MeOH: 5% acetic acid 50:50, inject
an 80 \xL aliquot.

HPLCVARIABLES
Column: 250 X 4.6 5 |xm Zorbax ODS C18
Mobile phase: MeCN: MeOH: water 44:11:45
Flow rate: 1
Injection volume: 80
Detector: UV 242; Radioactivity

CHROMATOGRAM
Internal standard: hydrocortisone 21-S-propionate (JO 498)
OTHER SUBSTANCES
Extracted: metabolites, budesonide

KEYWORDS
rat; lung; radiolabeled; pharmacokinetics; plasma
REFERENCE
Chanoine, F.; Grenot, C; Heidmann, P.; Junien, J.L. Pharmacokinetics of butixocort 21-propionate, bu-
desonide, and beclomethasone dipropionate in the rat after intratracheal, intravenous, and oral treat-
ments. Drug Metab.Dispos., 1991, 19, 546-553

SAMPLE
Matrix: formulations
Sample preparation: Ointment. Add pentane:EtOH 75:25 to ointment, sonicate for 20
min, dilute an aliquot to 100 mL with MeOH, allow to settle. Centrifuge and filter an
aliquot of the supernatant, inject an aliquot of the nitrate. Cream, lotion. Stir cream or
lotion in EtOH: THF: water 25:25:50 at 40° for 15 min, cool in an ice bath. Centrifuge
and filter an aliquot of the supernatant, inject an aliquot of the filtrate. Gel. Dissolve gel
in EtOH, sonicate, filter, inject an aliquot.

HPLCVARIABLES
Guard column: present but not specified
Column: 250 X 2.1 10 |xm Bondapak C18
Mobile phase: MeCN: water 48:52 containing 0.65% acetic acid, pH 3.18 (At the end of
each day flush guard column only with MeOH: THF 75:25 for 30 min.)
Flow rate: 1
Injection volume: 20
Detector: UV 251

CHROMATOGRAM
Retention time: 8.78

OTHER SUBSTANCES
Simultaneous: bamipine lactate, betamethasone-17-valerate, dexamethasone, hydrocorti-
sone-21-acetate

KEYWORDS
ointment; creams; lotions; gels

REFERENCE
Kountourellis, J.E.; Markopoulou, C.K.; Ebete, K.O.; Stratis, J.A. Separation and determination of some
corticosteroids combined with bamipine in pharmaceutical formulations by high performance liquid
chromatography. J.Liq.Chromatogr., 1995, 18, 3507-3517

SAMPLE
Matrix: ileostomy effluent
Sample preparation: Dilute ileostomy effluent 1:2 by weight with water and mix with
100 |xL 11 [LgImL 17-hydroxyprogesterone. Extract 3 g aliquot three times with 10 mL
dichloromethane by shaking for 1 min and centrifuging at 2000 rpm for 2 min. Wash
combined extracts successively with 2 mL 0.1 M NaOH and 4 mL water by shaking for
30 s and centrifuging for 1 min then dry the organic layer under air at 40°. Take up the
extract in 1 mL MeOH, add 1.1 mL water and apply to C18 Bond Elut SPE cartridge.
Wash with 10 mL water, wash with 5 mL MeOH: water 45:55, elute with 2 mL MeOH.
Add 50 |xL 20 \xg/mL progesterone to the eluate, dry at 40°, take up in 100 |xL MeOH,
inject 10 JJLL aliquot.

HPLC VARIABLES
Guard column: Bondapak C18/Corasil
Column: 300 X 3.9 ixBondapak C18
Mobile phase: MeOH: 50 mM pH 3.0 sodium phosphate buffer 55:45
Flow rate: 3
Injection volume: 10
Detector: UV 254; UV 238

CHROMATOGRAM
Retention time: 21.3
Internal standard: 17-hydroxyprogesterone (6.0), progesterone (11.6)

OTHER SUBSTANCES
Extracted: beclomethasone alcohol, beclomethasone 17-monopropionate

KEYWORDS
SPE

REFERENCE
Levine, D.S.; Raisys, V.A.; Ainardi, V. Coating of oral beclomethasone dipropionate capsules with cel-
lulose acetate phthalate enhances delivery of topically active antiinflammatory drug to the terminal
ileum. Gastroenterology, 1987, 92, 1037-1044

SAMPLE
Matrix: solutions

HPLC VARIABLES
Column: 250 X 4.6 Zorbax SAX
Mobile phase: MeOH: buffer 50:50 (Buffer was 180 mM Na2HPO4 adjusted to pH 3.00 ±
0.05 with 180 mM orthophosphoric acid. Pass mobile phase through a 250 X 4.6 25-40
ixm silica (HPLC Technology) column to saturate it with silica.)
Flow rate: 1
Detector: UV 253

CHROMATOGRAM
Retention time: 2.7

OTHER SUBSTANCES
Simultaneous: cromolyn, minocromil, nedocromil, quinoline yellow, saccharin, salicylic acid
Interfering: acetaminophen, albuterol, aspartame, aspirin, caffeine, isoproterenol, men-
thol, reproterol, riboflavin, sorbitan trioleate, terbutaline, theophylline

REFERENCE
Baker, P.R.; Gardner, J.J.; Wilkinson, D. Automated high-performance liquid chromatographic method
for the determination of nedocromil sodium in human urine using bimodal column switching.
J.Chromatogr.B, 1995, 668, 59-65

SAMPLE
Matrix: tissue
Sample preparation: 100 mg Tissue + 2 mL Ringer's pH 6.8 phosphate buffer + 2 mL
EtOH, centrifuge, wash residue twice. Pool supernatant and washings and evaporate to
dryness, take up in 400 (JLL EtOH, inject an aliquot.

HPLCVARIABLES
Guard column: present but not specified
Column: ixBondapak C18
Mobile phase: Gradient. MeOH.water 40:60 to 80:20, time not specified
Flow rate: 1.5
Detector: UV 254
OTHER SUBSTANCES
Extracted: beclomethasone monopropionate, beclomethasone, cyclomethasone

KEYWORDS
lung
REFERENCE
Ronca-Testoni, S. Hydrolysis of cyclomethasone by the human lung. Int.J.Clin.Pharmacol.Res., 1983, 3,
17-20

•— • •
ANNOTATED BIBLIOGRAPHY
Valvo, L.; Paris, A.; Savella, A.L.; Gallinella, B.; Ciranni Signoretti, E. General high-performance liquid
chromatographic procedures for the rapid screening of natural and synthetic corticosteroids.
J.Pharm.Biomed.AnaL, 1994, 12, 805-810 [gradient; reverse phase; normal phase; also betainetha-
sone, betamethasone 21-acetate, betamethasone 17,21-dipropionate, betamethasone 21-disodium
phosphate, betamethasone 17-valerate, cortisone, cortisone 21-acetate, 11-deoxycorticosterone 21-
acetate, dexamethasone, dexamethasone 21-acetate, dexamethasone 21-disodium phosphate, fluoci-
nolone, fluocinolone acetonide, 9a-fluorohydrocortisone, 9a-fluorohydrocortisone 21-acetate, 9a-fluo-
roprednisolone, 9a-fluoroprednisolone 21-acetate, hydrocortisone, hydrocortisone 21-acetate,
hydrocortisone 21-hemisuccinate, 6a-methylprednisolone, 6a-methylprednisolone 21-acetate, Ga-
rnet hylprednisolone 21-sodium succinate, prednisolone, prednisolone 21-acetate, prednisolone 21-di-
sodium phosphate, prednisolone 21-pivalate, prednisolone 21-sodium succinate, prednisone, triam-
cinolone, triamcinolone acetonide]
Girault, J.; Istin, B.; Malgouyat, J.M.; Brisson, A.M.; Fourtillan, J.B. Simultaneous determination of
beclomethasone, beclomethasone monopropionate and beclomethasone dipropionate in biological flu-
ids using a particle beam interface for combining liquid chromatography with negative-ion chemical
ionization mass spectrometry. J.Chromatogr., 1991, 564, 43-53 [plasma; urine; LC-MS; LOQ 1
ng/mL]
Benazepril
Molecular formula: C24H28N2O5
Molecular weight: 424.5
CAS Registry No.: 86541-75-5 (benazepril),
86541 -74-4 (benazepril hydrochloride)

SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform:
isopropanol: n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800
g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum
at 45°, reconstitute the residue in 100 JJIL mobile phase, centrifuge at 2800 g for 5 min,
inject a 50 fxL aliquot of the supernatant. (Buffer was saturated ammonium chloride
solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)

HPLCVARIABLES
Column: 300 X 3.9 4 |xm NovaPack C18
Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4
adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session
wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)
Column temperature: 30
Flow rate: 0.8
Injection volume: 50
Detector: UV 239

CHROMATOGRAM
Retention time: 5.01
Limit of detection: <120 ng/mL

OTHER SUBSTANCES
Extracted: acebutolol, acepromazine, aceprometazine, acetaminophen, aconitine, ajmaline,
albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alprenolol, amisulpride, am-
itriptyline, amodiaquine, amoxapine, aspirin, astemizole, atenolol, benperidol, benzo-
caine, benzoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine,
bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, carbinox-
amine, carpipramine, carteolol, celiprolol, cetirizine, chlorambucil, chlormezanone, chlo-
rophenacinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, ciben-
zoline, cicletanine, clemastine, clobazam, clomipramine, clonazepam, clonidine, clozapine,
cocaine, codeine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytara-
bine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextrome-
thorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihy-
dralazine, diltiazem, diphenhydramine, disopyramide, dosulepine, doxepin, doxylamine,
droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide,
floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen, fluvoxam-
ine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, histapyrrodine, hy-
droxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketam-
ine, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide,
loprazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, medifoxamine,
mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, me-
phentermine, mepivacaine, metformin, methadone, methaqualone, methocarbamol, meth-
otrexate, metipranolol, metoclopramide, metoprolol, mianserine, midazolam, minoxidil,
moclobemide, moperone, morphine, nadolol, nalbuphine, nalorphine, naloxone, naltrex-
one, naproxen, nialamide, nicardipine, nifedipine, niflumic acid, nimodipine, nitrazepam,
 nitrendipine, nizatidine, nortriptyline, omeprazole, opipramol, oxazepam, penbutolol, pen-
fluridol, pentazocine, phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide,
pindolol, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, pro-
guanil, promethazine, propafenone, propranolol, protriptyline, quinidine, quinine, quin-
upramine, ramipril, ranitidine, reserpine, ritodrine, secobarbital, sotalol, strychnine, sul-
finpyrazole, sulindac, sulpride, sultopride, suriclone, temazepam, tenoxicam, terbutaline,
terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, tianeptine,
tiapride, tiaprofenic acid, timolol, tioclomarol, tofisopam, tolbutamide, toloxatone, tria-
zolam, trifluoperazine, trifluperidol, trimipramine, triprolidine, tropatenine, verapamil,
viloxazine, vinblastine, yohimbine, zolpidem, zopiclone, zorubicine
Interfering: acenocoumarol, benazepril, chlordiazepoxide, clorazepate, dipyridamole, me-
tapramine, mexiletine, nomifensine, oxprenolol, pipamperone, pyrimethamine, ticlopi-
dine, trazodone, vincristine, vindesine, warfarin

KEYWORDS
whole blood; plasma

REFERENCE
Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic ScL,
1995, 40, 254-262

SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C2 SPE cartridge with 2 mL MeOH and 2
mL water. Adjust pH of plasma to 1.0 with dilute phosphoric acid, add 1 mL to the SPE
cartridge, wash with pH 1.0 dilute phosphoric acid, wash with water, wash with EtOH,
wash with MeCN, air dry, elute with 200 \xL mobile phase, inject an aliquot.

HPLCVARIABLES
Guard column: Newguard-Phenyl
Column: 150 X 3.9 ixBondapak phenyl
Mobile phase: MeOH: water 49:51 with 1 vial PIC-A reagent/L, adjusted to pH 7.0 with
phosphoric acid
Flow rate: 1
Detector: UV 210

KEYWORDS
plasma; dog; SPE; pharmacokinetics

REFERENCE
Kim, J.S.; Oberle, R.L.; Krummel, D.A.; Dressman, J.B.; Fleisher, D. Absorption of ACE inhibitors from
small intestine and colon. J.Pharm.ScL, 1994, 83, 1350-1356

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 Spherisorh 5 ODS-2
Mobile phase: n-Propanol: buffer 20:80 (Buffer was pH 3.0 phosphate buffer containing
0.4% triethylamine.)
Flow rate: 1
Detector: UV 240

CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Simultaneous: captopril, cilazapril, enalapril, quinapril, ramipril

REFERENCE
Barbato, F.; Morrica, P.; Quaglia, F. Analysis of ACE inhibitor drugs by high performance liquid chro-
matography. Farmaco, 1994, 49, 457-460
Betamethasone
Molecular formula: C22H29FO5
Molecular weight: 392.5
CAS Registry No.: 378-44-9, 987-24-6 (acetate), 22298-29-9
(benzoate), 5593-20-4 (dipropionate), 151-73-5
(sodium phosphate), 2152-44-5 (17-valerate),
5534-05-4 (acibutate), 360-63-4 (dihydrogen phosphate)

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL water containing 5 jxg/mL 2,3-diaminona-
phthalene and 3.5 |xg/mL 18-hydroxy-ll-deoxycorticosterone + 1 mL 250 mM NaOH + 7
mL diethyl ether, shake on a rotary shaker for 15 min, repeat extraction. Combine the
organic layers and evaporate them to dryness under a stream of nitrogen at 30-40°, re-
constitute the residue in 70 jxL MeOH: 100 mM perchloric acid 50:50, inject a 20 |xL
aliquot.

HPLCVARIABLES
Column: 150 X 3.9 4 |xm Nova-Pak C18
Mobile phase: Gradient. A was 58 mM NaH2PO4 containing 6 mM sodium heptanesulfon-
ate, adjusted to pH 3.1 with concentrated phosphoric acid. B was MeCN: MeOH 85:15.
A:B from 100:0 to 78:22 over 5 min, to 70:30 over 12 min, maintain at 70:30 for 4 min,
to 65:35 over 9 min.
Flow rate: 1
Injection volume: 20
Detector: UV 245; UV 256; UV 343

CHROMATOGRAM
Retention time: 19.45
Internal standard: 2,3-diaminonaphthalene (10.71), 18-hydroxy-ll-deoxycorticosterone
(15.85)
Limit of detection: 1-10 ng/mL (245 nm)

OTHER SUBSTANCES
Extracted: chloroquine, corticosterone, cortisone, dexamethasone, fluocinolone acetonide,
fluendrenolide, fhiorometholone, fluprednisolone, hydrocortisone, hydroxychloroquine,
178-hydroxyprogesterone, meprednisone, methylprednisolone, methylprednisolone ace-
tate, paramethasone, prednisolone, prednisone, progesterone, triamcinolone
Noninterfering: aspirin, ibuprofen, indomethacin, phenylbutazone, pregnenolone

KEYWORDS
serum

REFERENCE
Volin, P. Simple and specific reversed-phase liquid chromatographic method with diode-array detection
for simultaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids.
J.Chromatogr.B, 1995, 666, 347-353

SAMPLE
Matrix: blood
Sample preparation: Prepare a Sep-Pak Plus Environmental C18 cartridge by washing
with 15 mL MeOH then 15 mL water. 1 mL Serum + 200 |xL isopropanol: acetonitrile
1:1, mix, add to SPE cartridge, wash with 10 mL water, elute with 3 mL MeOH. Evap-
 orate the eluate at 50° under a stream of nitrogen, reconstitute in 200 |JLL mobile phase
A, inject a 20 |JLL aliquot.

HPLC VARIABLES
Guard column: jxBondapak C18 guard column
Column: 250 X 4.6 5 |xm Hypersil ODS
Mobile phase: Gradient. A was isopropanol: 50 mM pH 4.5 acetate buffer 10:90. B was
isopropanol:50 mM pH 4.5 acetate buffer 30:70. A:B from 90:10 to 30:70 over 25 min,
hold at 30:70 for 5 min, to 90:10 over 5 min, hold at 90:10 for 15 min before next
injection.
Column temperature: 40
Flow rate: 1
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 33
Internal standard: betamethasone

OTHER SUBSTANCES
Simultaneous: metabolites, cortisone, hydrocortisone, prednisolone, prednisone

KEYWORDS
serum; SPE; betamethasone is IS

REFERENCE
Hirata, H.; Kasama, T.; Sawai, Y.; Fike, R.R. Simultaneous determination of deflazacort metabolites II
and III, cortisol, cortisone, prednisolone and prednisone in human serum by reversed-phase high-
performance liquid chromatography. J.Chromatogr.B, 1994, 658, 55-61

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 5 mL dichloromethane, shake horizontally for 15
min, centrifuge at 1500 g for 15 min. Remove the organic layer and wash it with 100 |xL
100 mM NaOH then 1 mL water. Remove the aqueous phase and dry the organic phase
over 1 g of anhydrous sodium sulfate. Evaporate the organic phase to dryness under a
stream of nitrogen at not more than 37°, reconstitute in 200 jxL mobile phase, inject a
175 |xL aliquot.

HPLCVARIABLES
Guard column: 20 X 2 30-38 ynn HC Pellosil
Column: 250 X 4.6 5-6 p,m Zorbax SIL
Mobile phase: Heptane: dichloromethane: glacial acetic acid: ethanol 350:600:10:35
Flow rate: 2
Injection volume: 175
Detector: UV 254

CHROMATOGRAM
Retention time: 12
Internal standard: betamethasone

OTHER SUBSTANCES
Extracted: hydrocortisone, prednisolone, prednisone
Noninterfering: cyclosporin, ethinyl estradiol, ketoconazole, levonorgestrel, rapamycin,
tacrolimus, tenidap, tetrahydrocortisone
KEYWORDS
plasma; normal phase; betamethasone is IS

REFERENCE
Jusko, W.J.; Pyszczynski, N.A.; Bushway, M.S.; D'Ambrosio, R.; Mis, S.M. Fifteen years of operation of
a high-performance liquid chromatographic assay for prednisolone, cortisol and prednisone in
plasma. J.Chromatogr.B, 1994, 658, 47-54

SAMPLE
Matrix: blood
Sample preparation: Add 1 mL serum to a Sep Pak C18 SPE cartridge, wash with 4 mL
water, elute with 4 mL MeOH, evaporate to dryness under vacuum, reconstitute in 50
\xL MeCN: water 30:70, inject whole sample.

HPLCVARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere ODS
Mobile phase: MeCN: water 30:70
Flow rate: 1
Injection volume: 50
Detector: enzyme immunoassay of fractions

CHROMATOGRAM
Retention time: 16
Limit of detection: 0.3 pg

OTHER SUBSTANCES
Extracted: dexamethasone, flumethasone, triamcinolone
Noninterfering: endogenous steroids

KEYWORDS
serum; SPE; horse

REFERENCE
Friedrich, A.; Schulz, R.; Meyer, H.H. Use of enzyme immunoassay and reverse-phase high-performance
liquid chromatography to detect and confirm identity of dexamethasone in equine blood.
Am.J.Vet.Res., 1992, 53, 2213-2220

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 5 mL water + 1 mL 2 |xg/mL equilenin in MeOH
+ 50 |xL 0.1 M NaOH to adjust pH to 10, vortex briefly after each addition, shake with
10 mL dichloromethane for 10 min, centrifuge at 2000 g for 10 min. Wash organic layer
twice with 2 mL water, centrifuge 5 min, evaporate 8 mL of organic phase to dryness at
40° under a stream of nitrogen, reconstitute residue in 150 |xL mobile phase, inject 25 |JLL
aliquot.

HPLCVARIABLES
Column: 300 X 4 10 ^m jxBondapak C18
Mobile phase: MeOH: buffer 60:40 (Buffer was 10 mL 200 mM acetic acid + 15 mL 200
mM sodium acetate made up to 1 L, pH 4.8.)
Flow rate: 1
Injection volume: 25
Detector: UV 254

CHROMATOGRAM
Retention time: 5.5
Internal standard: equilenin (7.5)
Limit of detection: 5 ng/mL

OTHER SUBSTANCES
Simultaneous: deoxycortisol, hydrocortisone, prednisolone, prednisone, triamcinolone
Interfering: dexamethasone

KEYWORDS
Anal. Abs. 1982, 43, 4D182; plasma
REFERENCE
Bouquet, S.; Brisson, A.M.; Gombert, J. Dosage du cortisol et du 11-desoxycortisol plasmatiques par
chromatographie liquide haute performance [Cortisol and 11-desoxycortisol determination in blood
by high performance liquid chromatography]. Ann.Biol.Clin.(Paris), 1981, 39, 189-191

SAMPLE
Matrix: formulations
Sample preparation: Ointment. Add pentane:EtOH 75:25 to ointment, sonicate for 20
min, dilute an aliquot to 100 mL with MeOH, allow to settle. Centrifuge and filter an
aliquot of the supernatant, inject an aliquot of the nitrate. Cream, lotion. Stir cream or
lotion in EtOH: THF: water 25:25:50 at 40° for 15 min, cool in an ice bath. Centrifuge
and filter an aliquot of the supernatant, inject an aliquot of the filtrate. Gel. Dissolve gel
in EtOH, sonicate, filter, inject an aliquot.
HPLC VARIABLES
Guard column: present but not specified
Column: 250 X 2.1 10 |xm Bondapak C18
Mobile phase: MeCN: water 48:52 containing 0.65% acetic acid, pH 3.18 (At the end of
each day flush guard column only with MeOH: THF 75:25 for 30 min.)
Flow rate: 1
Injection volume: 20
Detector: UV 251

CHROMATOGRAM
Retention time: 5.17 (betamethasone-17-valerate)
OTHER SUBSTANCES
Simultaneous: bamipine lactate, beclomethasone dipropionate, dexamethasone, hydrocor-
tisone-21 -acetate

KEYWORDS
ointment; creams; lotions; gels
REFERENCE
Kountourellis, J.E.; Markopoulou, C.K.; Ebete, K.O.; Stratis, J.A. Separation and determination of some
corticosteroids combined with bamipine in pharmaceutical formulations by high performance liquid
chromatography. J.Liq.Chromatogr., 1995, 18, 3507-3517

SAMPLE
Matrix: formulations
Sample preparation: Pulverize tablets, weigh out amount equivalent to about 500 |xg be-
tamethasone, add 10 mL water, sonicate for 15 min, extract three times with 15 mL
chloroform: n-butanol 95:5. Combine extracts and filter them through 1 g anhydrous so-
dium sulfate moistened with chloroform: n-butanol 95:5. Collect filtrate and dilute it to
50 mL with chloroform: n-butanol 95:5. Remove a 1 mL aliquot, add 0.5 mL 40 JJLM cor-
tisone in mobile phase, mix, inject a 5 JJLL aliquot.
HPLCVARIABLES
Guard column: 5 |xm Guard-Pak Resolve Si (dead volume 60-75 uX.)
Column: 75 X 3.9 4 (xm Nova-Pak silica
Mobile phase: Dichloromethane: EtOH 34:1
Flow rate: 0.7
Injection volume: 5
Detector: UV 240

CHROMATOGRAM
Retention time: 6
Internal standard: cortisone (3)

OTHER SUBSTANCES
Simultaneous: dexamethasone, hydrocortisone, 6a-methylprednisolone, prednisolone,
prednisone

KEYWORDS
tablets; normal phase
REFERENCE
Liu, K.-R.; Chen, S.-H.; Wu, S.-M.; Kou, H.-S.; Wu, H.-L. High-performance liquid chromatographic
determination of betamethasone and dexamethasone. J.ChromatognA, 1994, 676, 455-460

SAMPLE
Matrix: formulations
Sample preparation: Triturate 1 tablet with a glass rod with 5 mL water, sonicate for 20
min, extract with 9 mL dichloromethane then three times with 5 mL dichloromethane,
filter (paper), make up to 25 mL with dichloromethane. Remove a 500 |xL aliquot, add
200 |xL 1.2 mM phenacetin in dichloromethane + 100 \xL 0.5 mM 4-dimethylaminopyri-
dine + 100 u,L 100 mM N-CBZ-Phe in dichloromethane + 100 JJLL 100 mM N, N'-dicy-
clohexylcarbodiimide in dichloromethane, shake mechanically at 30° for 1 h, inject a 10
IxL aliquot.

HPLCVARIABLES
Column: 75 X 3.9 4 |xm Nova-Pak silica
Mobile phase: n-Hexane: dichloromethane: isopropanol 100:100:4
Flow rate: 1
Injection volume: 10
Detector: UV 240

CHROMATOGRAM
Retention time: 7
Internal standard: phenacetin (10)
Limit of detection: 4.2 pmol

OTHER SUBSTANCES
Simultaneous: dexamethasone

KEYWORDS
tablets; normal phase; derivatization
REFERENCE
Chen, S.-H.; Wu, S.-M.; Wu, H.-L. Stereochemical analysis of betamethasone and dexamethasone by
derivatization and high-performance liquid chromatography. J.Chromatogr., 1992, 595, 203-208
SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 10 [xm Partisil 10 ODS
Mobile phase: MeOH: water 75:25
Flow rate: 1.2
Detector: UV 242

CHROMATOGRAM
Retention time: 5 (betamethasone 17-valerate)

REFERENCE
Mithani, S.D.; Bakatselou, V.; TenHoor, CN.; Dressman, J.B. Estimation of the increase in solubility of
drugs as a function of bile salt concentration. Pharm.Res., 1996, 13, 163-167

SAMPLE
Matrix: solutions
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 4 mL water then 3
mL MeOH. Add aqueous steroid solution to cartridge, elute with MeOH, inject a 20 \±L
aliquot.

HPLCVARIABLES
Column: 150 X 4 5 |xm Nucleosil C18
Mobile phase: MeCN: water 70:30
Flow rate: 1
Injection volume: 20
Detector: UV 239

CHROMATOGRAM
Limit of detection: 120 ng/mL

OTHER SUBSTANCES
Also analyzed: dexamethasone, flumethasone 21-acetate

KEYWORDS
SPE; for betamethasone 17-valerate

REFERENCE
Valenta, C; Janout, H. Corticosteroid analysis by HPLC with increased sensitivity by use of precolumn
concentration. J.Liq.Chromatogr., 1994, 17, 1141-1146

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 500 X 1 C18 (Alltech)
Mobile phase: MeOH: water 65:35
Flow rate: 0.04
Injection volume: 0.5
Detector: UV 254; MS, Hewlett Packard 5985, home-made interface (details in paper)

CHROMATOGRAM
Retention time: 21
OTHER SUBSTANCES
Simultaneous: metabolites, hydrocortisone

KEYWORDS
microbore

REFERENCE
Eckers, C; Skrabalak, D.S.; Henion, J. On-line direct liquid introduction interface for micro-liquid chro-
matography/mass spectrometry: application to drug analysis. Clin.Chem., 1982, 28, 1882-1886

SAMPLE
Matrix: solutions, tissue
Sample preparation: Buffer solutions. Condition a 3 mL Baker C18 SPE cartridge with
400 |xL MeOH. 100-500 |JLL 0.1 M pH 4.5 acetate buffer containing steroids + 100-1000
ng prednisone in MeOH, add to SPE cartridge, wash with 1 mL water, elute with 1'mL
MeOH. Evaporate the eluate to dryness under a stream of air, reconstitute in 40-200 JULL
dichloromethane: MeOH 98:2, inject a 40 |xL aliquot. Skin tissue. Crush tissue with 3 mL
MeOH: 100 mM pH 4.5 acetate buffer 20:80 using a Polytron tissue homogenizer, wash
homogenizer twice with the same solution, combine all solutions, add 400-4000 ng pred-
nisone in MeOH, add 10 mL dichloromethane, vortex for 1 min, centrifuge at 3000 rpm
for 5 min. Filter the organic phase through a column of Celite 545, evaporate to dryness
under a stream of air, reconstitute in 40-200 fxL dichloromethane: MeOH 98:2, inject a
40 JJLL aliquot.

HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrosorb Si-60
Mobile phase: n-Hexane: dichloromethane:MeOH: water 63.9:30:6:0.1
Flow rate: 1.2
Injection volume: 40-200
Detector: UV 240

CHROMATOGRAM
Retention time: 6.6
Internal standard: prednisone (5.3)
Limit of detection: 4 ng

KEYWORDS
SPE; normal phase

REFERENCE
Kubota, K.; Maibach, H.L In vitro percutaneous permeation of betamethasone and betamethasone 17-
valerate. J.Pharm.ScL, 1993, 82, 1039-1045

SAMPLE
Matrix: urine
Sample preparation: 3 mL Urine + 0.25 g NaCl, adjust pH to 9.0 with 0.5 g Na2HPO4,
add 4 mL dichloromethane, vortex 1 min, centrifuge at 3700 g for 3 min. Remove organic
phase and dry it over anhydrous sodium sulfate. Evaporate a 3 mL aliquot to dryness
under vacuum, reconstitute residue with 200 \xL 5 u,g/mL IS in MeOH, inject 20 u,L
aliquot.

HPLCVARIABLES
Column: 250 X 4.6 Hypersil ODS
Mobile phase: MeCN: water 32:68
Column temperature: 30
Flow rate: 1
Injection volume: 20
Detector: UV 245

CHROMATOGRAM
Retention time: 10
Internal standard: methylprednisolone (9)

OTHER SUBSTANCES
Extracted: corticosterone, cortisone, fluorocortisone, fluorocortisone acetate, hydrocorti-
sone, hydroxyprogesterone, prednisolone, prednisone, triamcinolone, triamcinolone
acetonide
Interfering: dexamethasone

KEYWORDS
SPE also discussed

REFERENCE
Santos-Montes, A.; Gonzalo-Lumbreras, R.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Solvent and solid-
phase extraction of natural and synthetic corticoids in human urine. J.Chromatogr.B, 1994, 652,
83-89

SAMPLE
Matrix: urine
Sample preparation: Dilute, if necessary, 100 u,L-l mL urine to 1 mL with water, add to
a Chem Elut high surface-area diatomaceous earth extraction column, after 5 min elute
with two 6 mL portions of ethyl acetate, combine the eluates and wash them twice with
1 mL 200 mM NaOH. Dry the organic layer over 1 g anhydrous sodium sulfate, evaporate
to dryness at 30° under a stream of nitrogen, reconstitute the residue in 250 |xL mobile
phase, inject a 50 |xL aliquot.

HPLCVARIABLES
Guard column: 70 X 6 37-53 \xm HC-Pellocil
Column: 250 X 4.6 5-6 |xm Zorbax SIL
Mobile phase: Dichloromethane:glacial acetic acid:MeOH 91.3:7.5:1.2
Flow rate: 2
Injection volume: 50
Detector: UV 254

CHROMATOGRAM
Retention time: 8.5
Internal standard: betamethasone

OTHER SUBSTANCES
Extracted: metabolites, hydrocortisone, 6p-hydroxycortisol, 20£-hydroxyprednisone, 6p-hy-
droxyprednisolone, 20a-hydroxyprednisolone, 20p-hydroxyprednisolone, prednisolone,
prednisone

KEYWORDS
betamethasone is IS; normal phase

REFERENCE
Garg, V.; Jusko, W.J. Simultaneous analysis of prednisone, prednisolone and their major hydroxylated
metabolites in urine by high-performanee liquid chromatography. J.Chromatogr., 1991, 567, 39-47
SAMPLE
Matrix: urine
Sample preparation: 3 mL Urine + 100 mg K2HPO4 + 500 mg anhydrous sodium sulfate
+ 5 mL diethyl ether, shake mechanically for 10 min, centrifuge at 2500 g for 5 min.
Remove the organic layer and evaporate it to dryness under vacuum, reconstitute the
residue in 200 |xL MeOH, filter (0.45 |xm), inject a 15 JJLL aliquot.

HPLCVARIABLES
Column: 60 X 4.6 3 |xm Hypersil ODS
Mobile phase: Gradient. MeOH: 150 mM ammonium acetate from 40:60 to 50:50 over 6
min, maintain at 50:50 for 1 min, to 60:40 over 3 min, maintain at 60:40 for 5 min
Flow rate: 0.8
Injection volume: 15
Detector: MS, Hewlett-Packard HP 5988A, vaporizer probe 92° decreased to 89° over 6 min,
decreased to 86° over 3 min, maintain at 86° for 5 min, ion source 276°, emission current
150 |JLA, electron energy 955 eV, positive ion mode, filament on

CHROMATOGRAM
Retention time: 7
Internal standard: betamethasone

OTHER SUBSTANCES
Extracted: corticosterone, cortisone, deoxycorticosterone, hydrocortisone, lla-hydroxypro-
gesterone, prednisolone, prednisone, triamcinolone, triamcinolone acetonide
KEYWORDS
betamethasone is IS
REFERENCE
Park, S.-J.; Kim, Y-J.; Pyo, H.-S.; Park, J. Analysis of corticosteroids in urine by HPLC and thermospray
LC/MS. J.Anal.Toxicol., 1990, 14, 102-108

• • •
ANNOTATED BIBLIOGRAPHY
Jonsson, G.; Astrom, A.; Andersson, P. Budesonide is metabolized by cytochrome P450 3A (CYP3A)
enzymes in human liver. Drug Metab.Dispos., 1995, 23, 137-142 [human; liver; microsomal incu-
bations; extracted budesonide; betamethasone is IS; SPE; gradient]
Garg, V.; Jusko, W. J. Effects of indomethacin on the pharmacokinetics and pharmacodynamics of pred-
nisolone in rats. J.Pharm.ScL, 1994, 83, 747-750 [rat; plasma; extracted corticosterone, predniso-
lone, prednisone; betamethasone is IS]
Santos-Montes, A.; Gonzalo-Lumbreras, R.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Extraction and
high-performance liquid chromatographic separation of deflazacort and its metabolite 21-hydroxy-
deflazacort. Application to urine samples. J.Chromatogr.B, 1994, 657, 248-253 [interfering triam-
cinolone acetonide; simultaneous corticosterone, cortisone, deflazacort, deoxycorticosterone,
dexamethasone, fludrocortisone, fludrocortisone acetate, hydrocortisone, 21-hydroxydeflazacort, l l a -
hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcinolone]
Santos-Montes, A.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Simultaneous determination of dexame-
thasone and betamethasone in pharmaceuticals by reversed-phase HPLC. Chromatographia, 1994,
39, 539-542 [simultaneous dexamethasone; methylprednisolone (IS); tablets; column temp 30; LOD
6 ng/mL; non-interfering corticosterone, cortisone, deflazacort, fludrocortisone, fludrocortisone ace-
tate, hydrocortisone, hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcin-
olone; interfering triamcinolone acetonide]
Valvo, L.; Paris, A.; Savella, A.L.; Gallinella, B.; Ciranni Signoretti, E. General high-performance liquid
chromatographic procedures for the rapid screening of natural and synthetic corticosteroids.
J.Pharm.Biomed.AnaL, 1994, 12, 805-810 [for betamethasone, betamethasone 21-acetate, betame-
thasone 17,21-dipropionate, betamethasone 21-disodium phosphate, betamethasone 17-valerate; gra-
 dient; reverse phase; normal phase; also beclomethasone, beclomethasone 17,21-dipropionate, corti-
sone, cortisone 21-acetate, 11-deoxycorticosterone 21-acetate, dexamethasone, dexamethasone
21-acetate, dexamethasone 21-disodium phosphate, fluocinolone, fluocinolone acetonide, 9a-fluoro-
hydrocortisone 21-acetate, 9a-fluorohydrocortisone, 9a-fluoroprednisolone, 9a-fluoroprednisolone
21-acetate, hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 21-hemisuccinate, 6a-methyl-
prednisolone, 6a-methylprednisolone 21-acetate, 6a-methylprednisolone 21-sodium succinate, pred-
nisolone, prednisolone 21-acetate, prednisolone 21-disodium phosphate, prednisolone 21-pivalate,
prednisolone 21-sodium succinate, prednisone, triamcinolone, triamcinolone acetonide]
Santos-Montes, A.; Gasco-Lopez, A.I.; Izquierdo-Hornillos, R. Optimization of the high-performance liq-
uid chromatographic separation of a mixture of natural and synthetic corticosteroids. J.Chromatogr.,
1993, 620, 15-23 [simultaneous corticosterone, cortisone, deoxycorticosterone, dexamethasone, fluo-
rocortisone, hydrocortisone, hydroxyprogesterone, methylprednisolone, prednisolone, prednisone,
triamcinolone]
Smith, E.W.; Haigh, J.M. In vitro diffusion cell design and validation. I. A stability-indicating high-
performance liquid chromatographic assay for betamethasone 17-valerate in purified isopropyl myr-
istate receptor phase. Pharm.Res., 1989, 6, 431-435
Maron, N.; Cristi, E.A.; Ramos, A.A. Determination of betamethasone 17-benzoate in lipophylic vehicles
by reversed-phase high-performance liquid chromatography. J.Pharm.Sci., 1988, 77, 638-639
Skrabalak, D.S.; Cuddy, K.K.; Henion, J.D. Quantitative determination of betamethasone and its major
metabolite in equine urine by micro-liquid chromatography-mass spectrometry. J.Chromatogr., 1985,
341, 261-269
Tokunaga, H.; Kimura, T.; Kawamura, J. Determination of glucocorticoids by liquid chromatography.
III. Application to ointments and a cream containing cortisone acetate, dexamethasone acetate, fluo-
rometholone, and betamethasone valerate. Chem.Pharm.BulL, 1984, 32, 4012-4016
Cairns, T.; Siegmund, E.G.; Stamp, J.J.; Skelly, J.P. Liquid chromatography mass spectrometry of dex-
amethasone and betamethasone. Biomed.Mass.Spectrom., 1983, 10, 203-208
Okumura, T. Application of thin-layer chromatography to high-performance liquid chromatographic sep-
aration of steroidal hormones and cephalosporin antibiotics. J.Liq.Chromatogr., 1981, 4, 1035—1064
[normal phase; also cephalexin, cephaloglycine, cephaloridine, cephalothin, cortisone, dexametha-
sone, hydrocortisone]
Petersen, M. C; Nation, R.L.; Ashley, J. J. Simultaneous determination of betamethasone, betamethasone
acetate and hydrocortisone in biological fluids using high-performance liquid chromatography.
J.Chromatogr., 1980, 183, 131-139
Bromocriptine
Molecular formula: C32H40BrN5O5
Molecular weight: 654.6
CAS Registry No.: 25614-03-3,22260-51-1 (mesylate)

SAMPLE
Matrix: blood, tissue
Sample preparation: Plasma. 1 mL Plasma + 10 JJLL 50 |xg/mL ergocriptine in MeCN:
MeOH:water 4:1:5 + 1 mL 2.5 M potassium carbonate, vortex, slowly add 4 mL MeCN
with vortexing for 30 s, centrifuge at 2200 g at -10° for 15 min, maintain at -10° for 30
min. Remove the organic layer and evaporate it to dryness, dissolve residue in 100 |JLL
MeCN:MeOH:water 4:1:5, filter (0.45 jxm), inject a 50 |xL aliquot. Tissue. Sonicate 300
mg rat brain tissue in 1 mL water (Heat Systems-Ultrasonics), add 10 LIL 10 |xg/mL
ergocriptine in MeCN: MeOH: water 4:1:5, add 1 mL 2.5 M aqueous potassium carbonate,
extract with 4 mL MeCN, centrifuge at 2200 g at -10° for 15 min, maintain at -10° for 30
min. Remove the organic layer and evaporate it to dryness, dissolve residue in 100 LLL
MeCN:MeOH: water 4:1:5, filter (0.45 Lim), inject a 50 |xL aliquot. (All glassware should
be silanized.)

HPLCVARIABLES
Column: 70 X 4.6 3 \xm Ultrasphere XL C8
Mobile phase: MeCN: isopropanol: 25.3 mM ammonium carbonate 40:6:54 (After each run
elute column with MeCN: water 80:20 for 15 min.)
Flow rate: 1.2
Injection volume: 50
Detector: UV 310

CHROMATOGRAM
Retention time: 10.5
Internal standard: ergocriptine (6)
Limit of detection: 19.5 ng/mL (plasma); 65 ng/g (tissue)

KEYWORDS
plasma; rat; brain

REFERENCE
Phelan, D.G.; Greig, N.H.; Rapoport, S.I.; Soncrant, T.T. High-performance liquid chromatographic assay
of bromocriptine in rat plasma and brain. J.Chromatogr., 1990, 533, 264-270

SAMPLE
Matrix: solutions

HPLC VARIABLES
Column: 250 x 4.6 5 ^m Supelcosil LC-DP (A) or 250 x 4 5 jjim LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer
was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust
pH to 3.4 with dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Injection volume: 25
Detector: UV 229

CHROMATOGRAM
Retention time: 10.80 (A), 6.21 (B)

OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,
albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atro-
pine, azatadine, baclofen, benzocaine, brompheniramine, brotizolam, bupivacaine,
buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine,
chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine,
chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride,
clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cy-
clobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, di-
phenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine,
doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen,
fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, fu-
rosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine,
hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine,
ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ie-
vorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic
acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,
methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyl-
dopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-
zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen,
nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazo-
line, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine,
pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,
phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, pra-
zosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, pro-
methazine, propafenone, propantheline, propiomazine, propofol, propranolol, protripty-
line, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone,
salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyra-
zone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethyl-
perazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin,
trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, tri-
methoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone

KEYWORDS
some details of plasma extraction

REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology.
J.Chromatogr.A, 1995, 692, 103-119

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 150 X 3.9 5 |xm Spherisorb C8
Mobile phase: MeCN: buffer 60:40 (Buffer was 1.5 mL triethylamine in 1 L water adjusted
to pH 3.0 with 85% phosphoric acid.)
Flow rate: 1.5
Injection volume: 200
Detector: UV 199

CHROMATOGRAM
Retention time: 5.5

OTHER SUBSTANCES
Simultaneous: benztropine mesylate, biperiden, desipramine, hyoscyamine, orphenadrine
Noninterfering: amantadine, carbidopa, levodopa

REFERENCE
Selinger, K.; Lebel, G.; Hill, H.M.; Discenza, C. High-performance liquid chromatographic method for
the analysis of benztropine in human plasma. J.Chromatogr., 1989, 491, 248-252
Buspirone
Molecular formula: C2IH31N5O2
Molecular weight: 385.5
CAS Registry No.: 36505-84-7 (buspirone),
33386-08-2 (buspirone hydrochloride)

SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform:
isopropanol.n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800
g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum
at 45°, reconstitute the residue in 100 jxL. mobile phase, centrifuge at 2800 g for 5 min,
inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride
solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)

HPLCVARIABLES
Column: 300 X 3.9 4 |xm NovaPack C18
Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4
adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session
wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)
Column temperature: 30
Flow rate: 0.8
Injection volume: 50
Detector: UV 236

CHROMATOGRAM
Retention time: 5.39
Limit of detection: <120 ng/mL

OTHER SUBSTANCES
Also analyzed: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen,
aconitine, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alpren-
olol, amisulpride, amitriptyline, amodiaquine, amoxapine, aspirin, astemizole, atenolol,
benazepril, benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bisoprolol, brom-
azepam, brompheniramine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine,
carbamazepine, carbinoxamine, carpipramine, carteolol, celiprolol, cetirizine, chloram-
bucil, chlordiazepoxide, chlormezanone, chlorophenacinone, chloroquine, chlorphenira-
mine, chlorpromazine, chlorpropamide, cibenzoline, cicletanine, clemastine, clobazam,
clomipramine, clonazepam, clonidine, clorazepate, clozapine, cocaine, codeine, colchicine,
cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytarabine, dacarbazine, daunorub-
icin, debrisoquine, demexiptiline, desipramine, dextromethorphan, dextromoramide, dex-
tropropoxyphene, diazepam, diazoxide, diclofenac, dihydralazine, diltiazem, diphenhy-
dramine, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol,
ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafen-
ine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen, fluvoxamine, gliben-
clamide, glibornuride, glipizide, glutethimide, haloperidol, histapyrrodine, hydroxychlor-
oquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine,
ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, lo-
prazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, medifoxamine,
mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, me-
phentermine, mepivacaine, metapramine, metformin, methadone, methaqualone,
methocarbamol, methotrexate, metipranolol, metoclopramide, metoprolol, mexiletine,
mianserine, midazolam, minoxidil, moclobemide, moperone, morphine, nadolol, nalbu-
phine, nalorphine, naloxone, naltrexone, naproxen, nialamide, nicardipine, nifedipine, ni-
 flumic acid, nimodipine, nitrazepam, nitrendipine, nizatidine, nomifensine, nortriptyline,
omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phen-
cyclidine, phenobarbital, phenol, phenylbutazone, pimozide, pindolol, pipamperone, pirox-
icam, prazepam, prazosin, prilocaine, procainamide, procarbazine, proguanil, prometha-
zine, propafenone, propranolol, protriptyline, pyrimethamine, quinidine, quinine,
quinupramine, ramipril, ranitidine, reserpine, ritodrine, secobarbital, sotalol, strychnine,
sulfinpyrazole, sulindac, sulpride, sultopride, suriclone, temazepam, tenoxicam, terbuta-
line, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, ti-
aneptine, tiapride, tiaprofenic acid, ticlopidine, timolol, tioclomarol, tofisopam, tolbuta-
mide, toloxatone, trazodone, triazolam, trifluoperazine, trifluperidol, trimipramine,
triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, war-
farin, yohimbine, zolpidem, zopiclone, zorubicine

KEYWORDS
whole blood; plasma

REFERENCE
Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic Sci.,
1995, 40, 254-262

SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL Baker carboxylic acid SPE cartridge with 5 mL 2
M HCl and 10 mL water. Mix 500 |JLL plasma + 5 mL water and add to column. Wash
with 5 mL water and elute with 2 mL 1 M formic acid. Evaporate eluate to dryness under
vacuum and dissolve residue in 200 |xL mobile phase, inject a 20 |JLL aliquot.

HPLCVARIABLES
Guard column: 5 |xm Cyanonitrile
Column: 33 X 4.6 Supelcosil LC-CN
Mobile phase: MeCN: 20 mM pH 7 potassium phosphate buffer 43:57 adjusted to pH 7.34
with 0.5 M KOH
Flow rate: 0.4
Injection volume: 20
Detector: E, ESA Coulochem model 5100 A, two porous graphite working electrodes and
associated palladium reference electrodes, +0.55 V for first electrode, +0.70 V for second
(monitoring) electrode

CHROMATOGRAM
Retention time: 10
Limit of detection: 1 ng/mL

OTHER SUBSTANCES
Extracted: metabolites, l-(2-pyrimidinyl)piperazine

KEYWORDS
plasma; mouse; SPE

REFERENCE
Betto, P.; Meneguz, A.; Ricciarello, G.; Pichini, S. Simultaneous high-performance liquid chromato-
graphic analysis of buspirone and its metabolite l-(2-pyrimidinyl)-piperazine in plasma using elec-
trochemical detection. J.Chromatogr., 1992, 575, 117-121

SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL C18 Supelco SPE cartridge with two 1 mL portions
of MeOH and two 1 mL portions of 50 mM KH2PO4 adjusted to pH 7.2 with 2 M NaOH,
add 2 mL serum and flush through 2-5 mL air. Wash with two 1 mL portions of 50 mM
KH2PO4 adjusted to pH 7.2 with 2 M NaOH, wash with two 1 mL and one 0.5 mL portions
of MeOH: water 1:1 and flush through with 2-5 mL of air. Elute with 1 mL MeCN:
triethylamine 99:1. Evaporate solvent at 37° under a stream of air and dissolve residue
in 100 IJLL of MeCN: 5 mM KH2PO4 + 0.1% triethylamine adjusted to pH 2.5 with ortho-
phosphoric acid 45:55, centrifuge at 5000 g for 10 min, inject a 70 |xL aliquot onto column
A with mobile phase A and elute to waste, switch a 0.7 min fraction onto column B
(retention time is ca. 3.6 min) and chromatograph on column B with mobile phase B,
monitor the effluent from column B.

HPLCVARIABLES
Column: A 150 X 4.6 5 jxm Spherisorb ODS2; B 150 X 4.6 5 fxm Spherisorb ODS2
Mobile phase: A MeCN: 5 mM KH2PO4 + 0.1% triethylamine adjusted to pH 2.5 with or-
thophosphoric acid 45:55; B MeCN: 5 mM KH2PO4 + 0.2% triethylamine adjusted to pH
2.5 with orthophosphoric acid 55:45, containing 5 mM sodium lauryl sulfate
Flow rate: 1.2
Injection volume: 70
Detector: UV 235

CHROMATOGRAM
Retention time: 7 (on column B)
Limit of detection: 0.2 ng/mL

KEYWORDS
serum; column-switching; heart-cut; SPE; pharmacokinetics

REFERENCE
Kristjansson, F. Sensitive determination of buspirone in serum by solid-phase extraction and two-di-
mensional high-performance liquid chromatography. J.Chromatogr., 1991, 566, 250-256

SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 50 |xL 400 ng/mL gepirone in EtOH + 0.5 mL 1 M
NaOH + 4 mL ethyl acetate, shake slowly mechanically for 10 min, centrifuge at 400 g
for 10 min. Freeze lower plasma layer in dry ice/acetone and decant ethyl acetate into a
centrifuge tube containing 2 mL 50 mM HGl, shake 10 min, centrifuge 400 g 10 min,
remove, discard organic phase. Add 500 |xL 4 M ammonia solution and 400 |xL butyl
acetate to the aqueous phase, mix for 15-20 sec, centrifuge at 400 g for 10 min. Remove
organic layer and evaporate it to dryness at 30° under vacuum. Reconstitute in 90 \xL
mobile phase, inject an aliquot.

HPLCVARIABLES
Guard column: 5 |xm Cyanonitrile (Anachem)
Column: 10 X 4.6 5 |xm cyanonitrile (Brownlee)
Mobile phase: MeCN: 40 mM potassium phosphate buffer adjusted to pH 6.6 with 2 M
KOH 34:66 (Columns were initially conditioned with 50 mL water; then 5 mM pH 4.8
sodium acetate buffer; then MeCN: 5 mM pH 4.8 sodium acetate buffer 40:60; then 5 mM
pH 4.8 potassium acetate; then mobile phase.)
Flow rate: 1.5
Injection volume: 50
Detector: E, ESA Coulochem model 5100 A, Model 5020 guard cell, guard cell 0.3 V, de-
tector 0.55 V for first electrode and 0.70 V for second electrode

CHROMATOGRAM
Retention time: 7.3
Internal standard: gepirone (5.4)
Limit of detection: 0.5 ng/mL

OTHER SUBSTANCES
Simultaneous: l-(2-pyrimidinyl)piperazine, metabolites, amitriptyline, chlorpromazine,
clomipramine, fluphenazine, imipramine
Noninterfering: caffeine, diazepam, desipramine, mianserin, zimeldine
Interfering: haloperidol

KEYWORDS
plasma

REFERENCE
Franklin, M. Determination of plasma buspirone by high-performance liquid chromatography with cou-
lometric detection. J.Chromatogr., 1990, 526, 590-596

SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 50 |xg/mL 1-phenylpiperazine + 1 mL pH
10 borate buffer + 5 mL chloroform: MeCN 8:2, agitate, centrifuge, repeat extraction.
Evaporate organic layers under reduced pressure below 40°, dissolve residue in 100 |xL
mobile phase, inject a 20 |JLL aliquot.

HPLCVARIABLES
Guard column: 23 X 3.6 37-50 |xm CN/Corasil
Column: 250 X 4.6 5 |xm Spherisorb CN
Mobile phase: MeOH: 5 mM KH2PO4 pH 7.4 35:65
Flow rate: 1.7
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 10
Internal standard: 1-phenylpiperazine (13)
Limit of detection: 5 ng/mL

OTHER SUBSTANCES
Extracted: l-(2-pyrimidinyl)piperazine, metabolites

KEYWORDS
plasma; rat

REFERENCE
Diaz-Marot, A.; Puigdellivol, E.; Salvatella, C; Cornelias, L.; Gassiot, M. Determination of buspirone
and l-(2-pyrimidinyl)piperazine in plasma samples by high-performance liquid chromatography.
J.Chromatogr., 1989, 490, 470-473

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 jjim LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer
was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust
pH to 3.4 with dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Injection volume: 25
Detector: UV 229

CHROMATOGRAM
Retention time: 9.07 (A)5 4.98 (B)

OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,
albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atro-
pine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bu-
pivacaine, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine,
chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlor-
propamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomi-
pramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclo-
benzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem,
diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine,
doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen,
fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, fu-
rosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine,
hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine,
ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ie-
vorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic
acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,
methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyl-
dopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-
zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen,
nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazo-
line, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine,
pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,
phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, pra-
zosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, pro-
methazine, propafenone, propantheline, propiomazine, propofol, propranolol, protripty-
line, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone,
salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyra-
zone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethyl-
perazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin,
trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, tri-
methoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone

KEYWORDS
some details of plasma extraction

REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology.
J.Chromatogr.A, 1995, 692, 103-119

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 Zorbax RX
Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL trie-
thylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL trie-
thylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over
30 min, maintain at 0:100 for 5 min.
Column temperature: 30
FIo1W rate: 2
Detector: UV 210

OTHER SUBSTANCES
Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline,
amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobar-
bital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzo-
tropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, bupre-
norphine, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal,
chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphe-
nesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine,
cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine,
colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide,
cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dex-
tromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylprorjion,
diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxy-
late, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol,
ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide,
etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fen-
proporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxy-
mesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glu-
tethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide,
hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen,
iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isopro-
terenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, Ii-
docaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclo-
fenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine,
mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesor-
idazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide,
methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldo-
pamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, meto-
prolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, na-
proxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid,
nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, ox-
ymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentaz-
ocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phency-
clidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine,
phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, pi-
perocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine,
propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, qua-
zepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol,
saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital,
strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine,
sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac,
tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, the-
obromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thiori-
dazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolme-
tin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine,
trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, ver-
apamil, vincamine, warfarin, yohimbine, zoxazolamine

REFERENCE
Hill, D. W.; Kind, A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J.Anal.ToxicoL,
1994, 18, 233-242
• « +
ANNOTATED BIBLIOGRAPHY
Gil, M.S.; Ochoa, C; Vega, S. High performance liquid chromatography of new potential anxiolytic drugs
and related benzodiazepines: A comparative study of hydrophobicity. J.Liq.Chromatogr., 1991, 14,
2141-2156 [also chlordiazepoxide, diazepam]
Sarati, S.; Guiso, G.; Spinelli, R.; Caccia, S. Determination of piribedil and its basic metabolites in
plasma by high-performance liquid chromatography. J.Chromatogr., 1991, 563, 323-332 [plasma;
rat; extracted piribedil; buspirone is IS; gradient]
Butalbital
Molecular formula: C11H16N2O3
Molecular weight: 224.3
CAS Registry No.: 77-26-9

SAMPLE
Matrix: blood
Sample preparation: 1 mL Blood + 1 mL water + 50 (xL 76 mg/L allobarbital in EtOH:
water 10:90 + 5 mL ethyl acetate, shake by hand, add 2 mL of 0.1 M HCl. Mix by
inversion with a mechanical shaker for 5 min. Centrifuge at 2700 rpm for 5-10 min.
Remove ethyl acetate and evaporate to dryness under a stream of nitrogen at room tem-
perature. Take up in 200 |xL MeOH, filter (0.45 |xm), inject a 20 |xL aliquot.

HPLCVARIABLES
Guard column: Guard-Pak precolumn insert
Column: 200 X 4.6 5 |xm Hypersil octadecylsilane
Mobile phase: Gradient. MeCN :1 mM pH 3.2 phosphate buffer from 20:80 to 40:60 over
10 min, stay at 40:60 for 6 min, to 20:80 over 4 min.
Column temperature: 60
Flow rate: 3
Injection volume: 20
Detector: UV 202

CHROMATOGRAM
Retention time: 5.0
Internal standard: allobarbital (2.9)
Limit of quantitation: 500 ng/mL

OTHER SUBSTANCES
Extracted: salicylic acid
Simultaneous: aspirin, caffeine

KEYWORDS
pharmacokinetics
REFERENCE
Drost, M.L.; Walter, L. Blood and plasma concentrations of butalbital following single oral doses in man.
JAnaLToxicoL, 1988, 22, 322-324

SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 200 jxL 50 jxg/mL hexobarbital in MeCN + 25 |xL
glacial acetic acid, vortex for 10 s, centrifuge for 1 min, inject a 30-100 |xL aliquot of the
supernatant.

HPLCVARIABLES
Column: (xBondapak C18
Mobile phase: Gradient. MeCN: 7.5 g/L NaH2PO4 adjusted to pH 3.2 with phosphoric acid
5:95 to 22:78 over 24 min, to 45:55 over 10 min, maintain at 45:55 for 5 min. Re-
equilibrate with 5:95 for 5 min.
Column temperature: 50
Flow rate: 3
Injection volume: 30-100
Detector: UV 210

CHROMATOGRAM
Retention time: 17.1
Internal standard: hexobarbital (20.6)
Limit of detection: 200-2000 ng/mL

OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, butabarbital, chlordiazepoxide, diazepam, eth-
chlorvynol, flurazepam, glutethimide, methaqualone, methyprylon, nitrazepam, pento-
barbital, phenobarbital, phenytoin, primidone, salicylic acid, secobarbital, theophylline
Simultaneous: amitriptyline, caffeine, clomipramine, codeine, desipramine, ethotoin, imip-
ramine, lidocaine, mesantoin, methsuximide, nirvanol, nortriptyline, oxazepam, procain-
amide, phenylpropanolamine, propranolol, quinidine

KEYWORDS
serum

REFERENCE
Kabra, P.M.; Stafford, B.E.; Marton, L.J. Rapid method for screening toxic drugs in serum with liquid
chromatography. J.AnalToxicoL, 1981, 5, 177-182

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 5 p,m Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer
was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust
pH to 3.4 with dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Injection volume: 25
Detector: UV 229

CHROMATOGRAM
Retention time: 5.77 (A), 5.12 (B)

OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,
albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atro-
pine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bu-
pivacaine, buspirone, butabarbital, caffeine, carbamazepine, cetirizine, chlorcyclizine,
chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlor-
propamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomi-
pramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclo-
benzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem,
diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine,
doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen,
fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, fu-
rosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine,
hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine,
ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ie-
vorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic
acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,
methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyl-
dopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-
 zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen,
nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazo-
line, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine,
pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,
phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, pra-
zosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, pro-
methazine, propafenone, propantheline, propiomazine, propofol, propranolol, protripty-
line, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone,
salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyra-
zone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethyl-
perazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin,
trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, tri-
methoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone

KEYWORDS
some details of plasma extraction

REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology.
J.Chromatogr.A, 1995, 692, 103-119

SAMPLE
Matrix: solutions
Sample preparation: Dissolve in mobile phase to a concentration of 50 jxg/mL.

HPLCVARIABLES
Column: 250 X 4 p-cyclodextrin polymer-coated silica (Chromatographia 1993, 36, 373)
Mobile phase: MeOH: water 50:50
Flow rate: 0.6
Injection volume: 20
Detector: UV 240

CHROMATOGRAM
Retention time: k' 1.15

OTHER SUBSTANCES
Simultaneous: amobarbital, aprobarbital, butabarbital, pentobarbital, phenobarbital, sec-
obarbital, thiopental

REFERENCE
Forgacs, E.; Cserhati, T. Retention behaviour of barbituric acid derivatives on a 3-cyelodextrin polymer-
coated silicon column. J.Chromatogr.A, 1994, 668, 395-402

SAMPLE
Matrix: solutions
Sample preparation: Dissolve in mobile phase at a concentration of 100 |xg/mL, inject a
5 |xL aliquot.

HPLCVARIABLES
Column: 300 X 2 ixBondapak C18
Mobile phase: MeCN: water 30:70 adjusted to pH 3.0 with formic acid
Flow rate: 0.27
Injection volume: 5
Detector: MS, VG TRIO 2000 single quadrupole MS with EI or CI; UV 270
CHROMATOGRAM
Retention time: 11.25

OTHER SUBSTANCES
Extracted: amobarbital, butabarbital, butethal, pentobarbital, talbutal

KEYWORDS
mass spectra given

REFERENCE
Ryan, T.W. Identification of barbiturates using high performance liquid chromatography-particle beam
EI/CI mass spectrometry. J.Liq.Chromatogr., 1994, 17, 867-881

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 300 X 3.9 ixBondapak C18
Mobile phase: MeCN: 10 mm KH2PO4 + 5 mM 1-decanesulfonic acid 30:70, adjusted to
pH 3.2 with 85% phosphoric acid
Flow rate: 1
Injection volume: 10
Detector: UV 214

CHROMATOGRAM
Retention time: 7.9
Internal standard: methyl paraben (7.0)
Limit of detection: 100 ng/mL

OTHER SUBSTANCES
Simultaneous: allobarbital, aprobarbital, barbital, mephobarbital, pentobarbital, pheno-
barbital, secobarbital, talbutal, vinbarbital

KEYWORDS
stability-indicating

REFERENCE
Ibrahim, RB. Simultaneous determination and separation of several barbiturates and analgesic products
by ion-pair high-performance liquid chromatography J.Liq.Chromatogr., 1993, 16, 2835-2851

• • •
ANNOTATED BIBLIOGRAPHY
Ryan, T.W. Resolution of the non-specific spectra of barbiturates by UV-photodiode array detection.
J.Liq.Chromatogr., 1993, 16, 315-329 [allobarbital, amobarbital, barbital, mephobarbital, pentobar-
bital, phenobarbital, secobarbital, talbutal, vinbarbital]